Hi Jenny,
It's true that a linear DNA marker is no use for sizing plasmids, however
there are ccc size markers available, I think Sigma sell one.
However you are also right to be concerned about damage to the plasmids
during isolation, however careful you are. This means that the same
plasmid DNA can be present in a preparation in all three forms: CCC ; OC
and linear.
I read a paper some years back that exploited the fact that UV
irradiation in the presence of EthBr generates nicks in CCC molecules, so
relaxing them. If I remember correctly, the sample was run through
agarose as normal, exposed to UV then electrophoresed at 90 degrees to
the first step, so that the original OC DNA, the newly formed OC DNA and
the still intact CCC form a little triangle. Bands outside the triangle
were assumed to be linear DNA.
Seems a lot of hassle so maybe the technology has improved since then.
The ref is Hinterman et al (1981) Plasmid 5 371.
Let me know if you can't get it and I'll try and dig it out.
Hope this helps,
Alastair
Alastair Hamilton
Institute of Aquaculture
University of Stirling
Scotland FK9 4LA
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---------- Forwarded message ----------
Date: 15 Oct 1998 13:24:23 GMT
From: ja.child at ukonline.co.uk
To: "bionet.microbiology mail newsgroup" <bionet-news at dl.ac.uk>
Subject: Sizing of E. coli plasmids- HELP!!
I'm getting in a bit of a mess over sizing of plasmids in wild-type isolates of E.coli.
Can someone wiser out there help me please?
Am I right in thinking that:
1. a linear DNA ladder can ONLY be used to size linear (e.g. digested) DNA fragments?
2. Undigested plasmids in E. coli can be sized by comparing the size with those from a standard marker strain such as the NCTC 50192 and 50193 in the UK. You would do this by plotting a calibration graph of log (plasmid MW in Md) against distance migrat
ed from the well, and read off the MW of the plasmids you want to size from this graph.
If this is the correct way of doing it, doesn't shearing of plasmids affect the result? (How can you tell from a plasmid gel whether this has happened?)
Can you accurately size plasmids this way, or should they ideally be linearised first?
Thanks,
Jenny